Process for the production of a new antibiotic sf-733 substance

ABSTRACT

A PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC SF-733 SUBSTANCE WHICH COMPRISES CULTIVATING SF-733 STRAIN OF STREPTOMYCES THERMOFLAVUS IN AN AUEOUS NUTRIENT MEDIUM UNDER SUBMERGED AEROBIC CONDITION AND RECOVERING THE ACTIVE INGREDIENT, AMORPHOUS FREE BASE OF SF-733 SUBSTANCE FROM THE MEDIUM AND MORE PARTICULARLY OBTAINING FREE BASE CRYSTAL OF SF-733 SUBSTANCE BY PASSING SAID AMORPHOUS SUBSTANCE THROUGH ETHANOL-SOLVATE STATE ON THE WAY OF CRYSTALLIZATION.

Mil-Rh 1974 TAKASHI SHOMURA ETAL' 3,7,842

PROCESS FOR THE PRODUCTION OF A NEW ANTIBIOTIC SIP- 3$ SUBSTANCEOriginal Filed Dec. 15, 1968 5 Sheets-Sheet 1 FIG.1

100mcg./ml. aqueous solution March 1974 TAKASHI SHOMURA ETAL 3,799,342

PROCESS FOR THE PRODUCTION OF A NEW ANTIBIOTIC SF- 33 SUBSTANCE 5Sheets-Sheet 2 Original Filed Dec. 5, 1968 oow 00m 000* com? 8 1. SM:002. 80w ooqw 00mm 8mm 8mm q A q d v d N OE March 6, 1974 TAKASHISHOMURA ETAL 3,799,842

PROCESS FOR THE PRODUCTION OF A NEW ANTIBIOTIC 51 53 SUBSTANCE OriginalFiled Dec. 3, 1968 5 Sheets-Sheet 5 Solvent A u u Eowc+mmw m u Eowc +mmwm mm. um

Solvent B m2. mm

1974 TAKASHl SHOMURA ETAL, Y 3,799,842

PROCESS FOR THE PRODUCTION OF A NEW AN'IIBIOTIC SF-733 SUBSTANCEOriginal Filed Dec. 5, 1968 5 'sheetssneet 4 v March 1974 TAKAsl-uSHOMURA ETAL 3,799,342

PROCESS FOR THE PRODUCTIOR OF A NEW ANTIBIOTIC SF 33 SUBSTANCE OriginalrFiled Dec. 5, 1968 5 Sheets-Sheet 5 United States Patent 3,799,842PROCESS FOR THE PRODUCTION OF A NEW ANTIBIOTIC SF-733 SUBSTANCE TakashiShomura and Norio Ezaki, Yokohama, Takashi Tsuruoka and Tomizo Niwa,Kawasaki, Eiichi Akita, Tokyo, and Taro Niida, Yokohama, Japan,assignors to Meiji Seika Kaisha, Ltd., Tokyo, Japan Original applicationDec. 3, 1968, Ser. No. 780,620, now Patent No. 3,661,829. Divided andthis application June 1, 1970, Ser. No. 54,058

Claims priority, application Japan, Dec. 18, 1967, 42/80,601; Oct. 15,1968, 43/74,634 Int. Cl. C12d 9/00 US. Cl. 195-80 R 4 Claims ABSTRACT OFTHE DISCLOSURE A process for the production of an antibiotic SF-733substance which comprises cultivating SF-733 strain of Streptomycesthermoflavus in' an aueous nutrient medium under submerged aerobiccondition and recovering the active ingredient, amorphous free base ofSF-733 substance from the medium and more particularly obtaining freebase crystal of SF-733 substance by passing said amorphous substancethrough cthanol-solvate state on the way of crystallization.

This application is a division of application Ser. No. 780,620 in thename of 'Shomura et al., filed Dec. 3, 1968, now US. Pat. No. 3,661,829.

This invention relates to a process for the production of a newantibiotic called SF-733 substance which is obtained by cultivating aspecific strain selected from the genus Streptomyces.

3,799,842 Patented Mar. 26, 1974 ful growth preventing action againstmicro-organisms in wide range such as gram positiveandnegativepathogenic bacteria and acid-fast bacteria, is produced in aculture broth of a specific strain belonging to the genus Streptomycesand its active substance may be recovered from the culture broth. Wehave called this active substance SF-733 substance. In this invention, astrain of Streptomyces which has an activity for producing SF-733substance in the culture broth in such an extent as is enough to berecovered is employed.

The SF-733 substance-producing microorganism was isolated by the presentinventors from a sample of soil collected in Tsu city, MIE Prefecture,Japan, and designated Streptomyces thermoflavus SF-733 which has beendeposited in the American Type Culture Collection, Washington, DC, underATCC No. 21294.

Referring to the drawings:

FIG. 1 is ultra-violet spectrum of amorphous SF-733 substance.

FIG. 2 is infrared spectrum of amorphous SF-733 substance (KBr tablet).

FIG. 3 is paper chromatogram of amorphous SF-733 substance and itsrelated antibiotics.

FIG. 4 is ultraviolet spectrum of free base crystals of SF-733substance.

FIG. 5 is infrared spectrum of free base crystal of SF- 733 substance.

Streptomyces thermoflavus SF-733 has the following characteristics:

(1) Microscopic observation:

(1) Aerial mycelium open spiral formed abundantly. (2) Spore: oval toelliptical, surface structure is spiny,

size is 0.8-1.0 x 1.1-1.4 micron.

We have found that an antibiotic which shows a power- (II)Characteristics on various culture media:

Culture medium Growth Aerial mycelium Soluble pigment Sucrose-Czapekagar (incubated at 28 Thick, good, wrinkled, penetrating into Veryscant, light yellowish white None.

0.). medium. Yellowish cream color, reverse light brownish cream color.Glycerol-Czapek agar (incubated at 28 Thick, good, wrinkled, penetratinginto Scant. White to cream color Do.

0. medium, light yellowish brown.

Krrinsky's glucose asparagine agar Good, penetrating into medium. DarkScant. Cream to light yellowish white Do.

(incubated at 28 0.).

color.

Ushinskys glucose asparagine agar Good, penetrating into medium,brownish yellow color (with greenish tinge).

(incubated at 28 0.).

cubated at 28 C.)

Starch synthetic agar (incubated at 28 Good, penetrating into medium.Yellow 0.). central part of colony shows brown (with greenish tinge).

Bouillon agar (incubated at 28 C.) Colonial, penetrating into medium,dark cream color, reverse brownish yellow color.

Abundant, powdery. Greenish yellow, Slight yellow.

gradually turns from grayish tinge to grayish brown. Greenish yellowpatches formed. Scent. White None.

None or scant, light yellow color Do.

Abundant, powdery, greenish yellow Slight yellow.

gradually turns to grayish brown 0 or. None or scant, light yellow colorNone.

cream color, reverse brownish yellow color. GllgOSQ bouillon agar(incubated at 28 Fine wrinkled. Yellowish brown color Scent, cream colorDo. Potato plug (incubated at 28 C.) Elevated, 1fine wrinkled, lightbrownish Scant, whitish gray color Do.

cream co or. Carrot plug (incubated at 28 C.) Colonial, dark creamcolor- None Do. Tyrosine agar (incubated at 28 C.) Deeply penetratinginto medium, dark Gray Do.

cream co or. Egg (incubated at 37 C.) Colonial, light brownish yellowcolor None Do. Loefiflg e r Ccgagulated serum (incubated Dark brownishyellow color do Do.

a Ba:t)o nitrate broth (incubated at 28 Sedimented to bottom colorlessdo Do. Skim milk (incubated at 37 C.) Yellowish orange ring on surfacein contact .....do Light brownish orange with glass, sedimented mass onbottom. color. Reaction of medium: pH 5.5. Gelatine stab (incubated at20 C.) Colorless to cream colondo None.

Cellulose medium (incubated at 28 C.) No growth 3 (III) Physiologicalproperties:

Production of hydrogen sulfide Negative. Production of tyrosinase Do.Production of nitrite Positive. Coagulation of skimmed milk Negative (28C.,

37 C.). Peptonization of skimmed milk Positive (37 C.);

negative (37 C.). Hydrolysis of starch Positive. Liquefaction ofgelatine Negative. Dissolution of Loeifers coagulated Positive (weak,

serum. 37 C.).

(IV) Utilization of carbon sources:

(Pridham-Gottliebs basal medium, incubated at 27 C.)

(1) Utilize: arabinose, rhamnose, glucose, mannose, galactose,saccharose, lactose, raflinose, dextrin, Inulin, starch, glycerol,sorbitol, mannitol, maltose.

(2) Doutful: inositol, sodium citrate.

(3) Not utilize: xylose, fructose, dulcitol, sodium acetate,

sodium succinate, salicin, cellulose.

(V) Optimum temperature: 40 C. (measured with temperature gradientincubator) As described above, the characteristics of the strain SF- 733can be summarized as follows:

Spiral formation in aerial mycelium; spore with spiny structure; creamto yellowish brown growth on synthetic media, yellowish green to grayishbrown aerial mycelium, lack of soluble pigment in general but lightyellow pigment on glucose-asparagine agar (Ushinsky) and starchsynthetic agar cream to yellowish brown growth, thin aerial mycelliumand no soluble pigment on organic media. The most distinctcharacteristic of SF-733 strain is that optimum temperature for growthis rather high as 40 C.

From the characteristics described above, SF-733 strain is deemed toclosely relate to flavus series of genus Streptomyces. Namely among thisseries, Streptomyces flwvus, Streptomyces flaveolus, Streptomycesflavovireus Streptm myces flavogrz'seus, Streptomyces alboflavusStreptomyces panvus, Streptomyces parvullus, Streptomyces kanamyceticus,Streptomyces griseoflavus and Streptomyces cellwloflavus may bementioned as closely related strains. Among these species, Streptomyceskanamycetius, Streptomyces flavorgriseus, Streptomyces alboflavus andStreptomyces celluloflavus are clearly differentiated from SF-733 strainbecause aerial mycelia of the former form no spiral while one of thelatter form abundant typical open spirals. Streptomyces parvullus whichforms smooth type spore is different from SF-733 strain spore surface ofwhich is spiny. Streptomyces parvus and Streptomyces griseoflavus areclearly ditferent from SF-733 strain because the former well grows oncellulose medium. Streptomyces flavus, Streptomyces flaveolus andStreptomyces flavovirens which may be said to be typical strains offlavus series are most closely related to SF-733 strain in respect ofvarious characteristics on various culture media.

These three organisms, however, are different from SF-733 strain in thefollowing respects. Namely Streptomyce s flaveolus the spores of whichare hairy surface is differentiated from SF-733 strain the spores ofwhich are spiny. The aerial mycelia of Streptomyces flavus andStreptomyces flavovireus are principally straight but in some cases formopen spirals while the aerial mycelium of SF-733 strain forms abundantopen spirals. In addition thereto Streptomyces flavovireus producesgreenish yellow soluble pigments on synthetic agar medium while SF-7 33strain does not produce soluble pigment. Further, Streptomyces flavusdoes not reduce nitrate and its optimum temperature for cultivation is25 C. while SF-733 strain produces nitrate and its optimum culturaltemperature is rather high as 40 C. As described above,

SF-733 strain is clearly different from above three organisms in respectof various important characteristics.

New antibiotic SF-733 substance produced by the SF-733 strain is atypical water-soluble basic antibiotic of wide range of anti-bacterialspectrum, and its optical rotation is Among the known antibiotics,neomycin, paromomycin, kanamycin, gentamicin and tenemycin and the likebelong to the same group. The strain SF-733 should he, therefore,compared with above antibioticproducing strains with respect to theirmycological relationship. Two gentamicin-producing strains are,Micromonospora echinospom and Micromonospora purprea. They do not belongto Streptomyces so that they are clearly different from SF-733 strain.Further neomycinproducing strains: Streptomyces fradz'ae, Streptomy'cesalbogriseolus, paromomycin-producing strain: Streptomyces rimosus formaparomomycinus, kanamycin-producing strain: Streptomyces kanamyceticus,tenemycin producing strain: Streptomyces tenebrarius are clearlydifferent from the SF-733 strain in respect to their morphologicalproperties as shown in following table and therefore they are differentorganisms from each other.

Comparison between SF-733 strain and the known species of strepto' myceswhich produce dextrorotary water-soluble basic antibiotics 1 SeventhInter-science Conference on Antimicrobial Agent and Chemotherapy, 2527October 1967 in Chicago.

As the result of above mycological investigation, we have confirmed thatSF-733 strain is a new micro-organism which produces water-soluble basicantibiotic and that in view of taxonomical standpoint it is a novelstrain of flavus series and have named it Stroptomyces thermoflavus nov.sp. (The properties of the compared species of Streptomyces based uponWaksman: The Actinomycetes, vol. 2, 1961.)

The characteristics of SF-733 strain are liable to vary as is observedwith other Streptomyces. Namely, artificial variants and mutants ofSF-733 may be obtained by various known mutagens such as ultra-violetrays, X-ray, high frequency wave, radioactive ray and chemicals. Allnatural and artificial variants and mutants which belong to Streptomycesthermoflavus and have activity producing SF-733 substance may beutilized in this invention.

According to the present invention, SF-733 strain may be cultured in aculture medium containing the nutrients which would be used incultivation of used microorganisms. For the nutrients, the knownnutrients which are utilized in cultivation of Streptomyces can be used.For example, glucose, starch, glycerol, dextrin, sucrose and the likeare useful as the carbon source. Soybean meal, wheat-embryo,meat-extract, peptone, dried yeast, corn steep liquor, distillerssoluble, ammonium sulfate, sodium nitrate and the like are useful as thenitrogen source. If necessary, inorganic salts such as calciumcarbonate, common salt, potassium chloride, phosphate may be added. Inaddition, organic and inorganic materials which aid the growth of SF-733strain and facilitate the production of SF-733 substance can be alsoadded. For the cultivation of SF-733 strain, liquid cultivation, inparticular, under submerged aerobic condition is most preferable.Suitable fermentation temperature is 25-40" C. mostly near 35 C. Theproduction of SF733 substance comes to maximum for 2-5 days in either ofshaking culture-and tank culture.

For the assay of SF-733, mycinassay agar (pH 7.8) may be used as mediumand Bascillus swbtilis ATCC No. 6633 as a test organism.

Linear relation between logarithm of concentration of SF-733 substanceand diameter of inhibitory ring against test organism was observed atthe level of 1-25 mcg./ml. and the values of diameters of inhibitoryring at such condition was 15-25 mm. respectively. (up-plate method).

SF-733 substance is a water soluble basic antibiotic as is clear fromthe physico-chemical properties described below and may be recoveredfrom the cultured broth by every known methods which are generallyavailable for the recovery of the known water soluble basic antibioticssuch as kanamycin, neomycin, etc.

SF-733 substance may be more easily adsorbed on active carbon atalkaline side and eluted more efiiciently with an aqueous alcohol oraqueous acetone at acidic pH.

SF-733 substance may also be purified With ion exchange resin as anadsorbent. For ion-exchange resin, for example, cation exchange resinsuch as Amberlite IRC-50 (Rohm & Haas) in NIL- Na+- or H+-type mayadvantageously be used. The elution is usually made with an aqueousacid, alkali or salt solution. Further, SF-733 substance may also beefiiciently deposited in the form of acid salt by adding watermiscibleorganic solvent to an aqueous solution of SF-733 substance acid salt.

A crude powder of SF-733 substance thus obtained can be further purifiedby ion-exchange chromatography with Dowex 1 x 2 (OH' type) (DowChemicals) or Amberlite CG-SO (NH type) (Rohm & Haas), whereupon whiteamorphous powder of free base of SF-7 33 substance can be obtained thepurity and unity of which was ascertained by paper chromatography, highvoltage paper electrophoresis and thin layer chromatography if itsN-acetyl derivative. The amorphous free base of SF-733 substance isstable in neutral and alkali side but is barely stable in acid side.

Physico-chemical properties of SF-7 33 substance are here described.Amorphous free base of SF-733 substance decomposes with eifervescence at178-185 C. and not shows a definite melting point. Said free base iseasily soluble in water, a little soluble in methanol and insoluble inorganic solvent such as acetone, butanol, ethyl acetate, benzene,hexane, ether. In electrophoresis, it moves at pH 1.8 towards cathodeand no existence of acidic group is detected from titration curve. Thesefacts prove that said free base is a basic substance. Its ultravioletabsorption spectrum is shown in FIG. 1. No specific absorption isobserved. Its infrared absorption spectrum is shown in FIG. 2. Anabsorption band which is peculiar to amino-sugar antibiotics isobserved.

The specific optical rotation of SF-733 substance (amorphous free base)in aqueous solution ((1) is +42". The reactions of ninhydrin, Molischand anthrone are positive, and the reactions of Fehling, Benedict,ferric chloride, maltol, biuret, Tollens and Sakaguchi are negaopticalrotation (in aqueous solution),

deg. Literatures +123 Umezawa at 9.1., Index of Antibiotics fromAetinomycetes Substances SF-733 Neomycin:

Hygromycin 13 Actinospectacm. Kasugamycin Capreomycin II +121 Kondo,Journal of Antibiotics series B. (1961) +13 do +126 +146 Umezawa et :11.Index of Antibiotics from Aetinomycetes +7 +6 Urnezawa et al., Index ofAntibiotics from Actinomycetes tive. The molecular weight estimated bythe vapour pressure equillibrium method is 475 which is sustained by thevalue (452 as tetra acid base) estimated from the titration curve.

The elemental analysis of a sample of SF-733 substance which was driedon phosphorous pentoxide in vacuo at 110 C. for 20 hrs. is as follows(percent): C, 44.16, H, 7.55, N, 11.92, 0, 36.21 and therefore themolecular formula is C H N O (theoretical value (percent): C, 44.93, H,7.54, N, 12.33, 0, 35.20, molecular weight: 454.49). It has been foundfrom the studies on chemical structure that SF-733 substance has thefollowing chemical structure of O-B-D-ribofuranos'ye-(l-5)-O-[a-2,6-diamino-2, 6-dideoxy D glucopyranosyl-(1-4)]-2-deoxystreptamine OH OH By descending paper chromatography usingn-butanolpyridine-acetic acid-"water (6:4:113, development for 4 days)and n-butanol saturated with water containing 2% p-toluene sulfonic acid(development for 25 hours), coloration by ninhydrin reaction andbioautography using Bacillus subtilis SF-733 substance shows in eachsolvent systems a single spot at 8 cm. and 14.8 cm. from original pointrespectively. In high voltage paper electrophoresis (3300 v., pH 1.8, 15minutes) also SF-733 substance moves 15 cm. to the cathode and shows asingle spot. Further on thin layer chromatography of silica gel using asa developer t-butanol-acetic acid-water (2: 1 :1), n-butanol-aceticacid-water (321:1) and n-butanol-pyridinewater (6:4:3) and aqueousmethanol, N-acetyl derivative of SF-733 substance shows a single spot atRf 0.40, 0.16, 0.50 and 0.70 respectively. This fact also clearly provesthe purity and unity of SF-733 substance.

As it is clear from above explanation, *SF-733 substance is one of watersoluble basic antibiotics and the optical rotation thereof isdextro-rotatory. Among the known antibiotics neomycin, paromomycin,kanamycin, gentamicin, destomycin, actinospectacin are to be comparedwith SF-733 substance.

SF-733 substance are compared with these known antibiotics in respect oftheir optical rotation (Table 1) and paper chromatography (Table 2 andFIG. 3) as follows.

TABLE 1 Page (Tokyo University Publishing Society, 1967).

(Tokyo University Publishing Society, 1967).

(Tokyo University Publishing Society, 1967).

TAB LE 2 Solvent B Solvent A Having Moving distance distance from fromoriginal original point point Substances (cm.) R 733 (am) R 733 SF-733substance base 8. 1 14. 8 1 Kauamycin:

7. 7 0. 96 19. 2 1. 30 3. 4 0. 43 17. 6 1. 18 3. 5 0.41 17. 5 1. 18Paromomycin:

I base 4. 1 0.56 10.8 0. 73 II base 4. 6 0. 58 10.0 0. 74 Gentamicin 0base 12. 0 1. 50 25.0 1. 69

1 Ratio of moving distance on the basis of SI -733 substance.

Nora-Solvent A: n-butanol saturated with water containing 2%p-toluenesuli'onio acid, descending method, development for 25 hours.Solvent B: n-butanol-pyridine-acetic acid-water (6:4:1z3), descendingmgtlziod, development for 4 days. Assay: Bioautography using Bacillus an118.

SF-733 substance may be clearly differentiated from kanamycin group (A,B, C), gentamicin group (A, C C neomycin A, destomycin A, B, hygromycinB, actinospectacin, kasugamycin and capreomycin with respect to theirspecific optical rotation in Table l and from neomycin B, C, paromomycinI, II, gentamicin C and kanamycin A, B with respect to Rt value on paperchromatograrn in Table 2 and FIG. 3. Further, SF-733 substance may beclearly differentiated from capreomycin II which has ultravioletabsorption maximum, from actinospectacin by analytical value ofnitrogen, from destomycin A, B and hygromycin B by characteristics oftheir antibacterial spectrum and their higher toxicity. FurthermoreSF-733 substance may be clearly differentiated from tenemycin (Seventh'Interscience Conference on Antimicrobial agent and Chemotherapy, 1967,from Oct. 25 to 27 in Chicago) because [a] value of N-acetyl derivativeof tenemycin is (+95-+130) while that of N-acetyl derivative of SF-733substance is +40. Thus, it is clear from above comparison that SF-733substance is a new antibiotic which does not coincide with any knownantibiotics.

The antibacterial spectrum of SF-733 substance against variousmicroorganisms is as follows.

Minimum inhibitory concentration by broth dilution method Minimuminhibitory concentration Culture Test orgamsms (meg/ml.) medium Bacillussubtilis ATCC 6633 0. 39 Bouillon medium. Bacillus cercus 6. 25 Do.Staphylococcus aureus 209-P.-- 3. 125 Do. Staphylococcus aureus 52-34..-3. 125 Do. Staphylococcus aurcus 193 3. 125 Do. 3 r aureus Smith 0. 39Do. Staphylococcus aurcus Terajima..- 0.19 Do. Sarcina lutea 100.0 D0.er L cor 12. 5 Do. Escherichia coli IAM 1253... 12.5 Do. Escherichiacoli IAM 1239... 12. 5 Do. Escherichia coli K-12 3.125 D0. Escherichiacoli chloramphenicol resistant. 1. 56 Do. Escherichia coli streptomycinresistant 12. 5 Do. Escherichia coli streptothricin resistant. 50 Do.Escherichia coli kanamycin resistant-. 100 Do. Shigella dysenteriae 5.25 D0. Shiyella jfexneri (streptomycin tetracyclin) 12.5 Do.

resistant. Salmonella typhi 3. 125 D0. Salmonella typhi paratyphi A- 12.5 Do. Salmonella typhi paratyphi B.-. 12.5 Do. .Klcbsiclla pneumoniae 6.25 Do. Proteus vulaaris 25. 0 D0. Pseudomtmas aeruginoaa 100. 0 Do.Mycobacterium smegmates 60 12.5 Glycerine bouillon medium. Mycobacteriumphlei 6- 25 0- C 'rr- 100.0 Sabouraub. Torula utilis 100. 0 D0.Saccharomyces cerevisi 100. 0 Do. Aspergillus niger 100 0 Do.

As it is clear from above table, SF-733 substance is a wide range activesubstance which are active to gram positive bacteria, gram negativebacteria and acid fast bacteria.

According to tests of acute toxicity by intravenous injection to mice,toxicity of SF-733 substance is very low such as LD is 1000 mg./kg. ofits free base and 500 mg./kg. of its sulfate and no abnormal progresswere observed after injection.

Amorphous free base of SF-733 substance may be crystallized by followingmethod.

Amorphous free base is dissolved in water and the solution isconcentrated to dryness while adding ethanol and distilling out water byazeotropic distillation, or the solution is first concentrated to syrupand then ethanol is added to form precipitate and concentrated todryness while further adding ethanol and distilling out water byazeotropic distillation to obtain white solid SF-733 substance in theform of ethanol-solvate. This white solid ethanol-solvate like SF-733substance is characterized by its higher solubility in methanol thancompletely dehydrated amorphous free base of SF-733 substance andcrystallized free base of SF-733 substance mentioned below.

The white solid ethanol-solvate like SF-733 substance thus obtained isdissolved in appropriate amount of methanol and left it alone wherebycrystals are quickly crystallized out. The desired free base crystal ofSF-733 substance may 'be obtained by, for example, recovering thecrystals by filtration, washing with a little methanol drying at roomtemperature in vacuo and further drying in vacuo in a glass desiccatorat 60 C. for 19 hours using phosphorus pentoxide as a drying agent, toremove methanol adhered to crystals.

The powder obtained by freeze-drying an aqueous solution of amorphousfree base of SF-733 substance and made absorb a suitable amount of waterhas a higher solubility in methanol as above mentioned white solidethanol-solvate but even when this powder is dissolved in methanol andleft alone, only a small amount of precipitate is formed but no crystalsof free base of SF-733 substance can be obtained. Thus forcrystallization of free base of SF-733 substance ,9 the present processwherein the material to be crystallized is caused to pass throughethanol-solvate state on the way to crystallization is essential.

The free base crystal of SF-733 substance is white needles or short rodsand fuses While decomposing with eifervescence at 192-l95 C.

The elemental analysis (percent): C, 44.19, H, 7.39, N, 11.60, 0 (bydifference), 36.82. Molecular weight estimated by the vapor pressureequilibrium method in an aqueous solution is 440 and one estimated bythe titration method in an aqueous solution is 470. Then the molecularformula of said free base crystal of SF-733 substance is C17H34N4Om(theoretical value (percent): C, 44.93, H, 7.54, N, 12.33, 0, 35.20,molecular weight, 454.49) and has the chemical structure ofO-p-D-ribofuramosyH1 5)- O[a-2,6-diamino-2,6-dideoxy-D-glucopyranosyl-(1 2-deoxystreptamine asmentioned above. Namely free base crystal of SF-733 substance is thesame compound as amorphous free base of SF-733 substance, and they aredifferent from each other only in the point that they are in differentexisting states.

The free base crystal of SF-733 substance is dextrorotatory and [04thereof is +42 (c.=1, H O). An aqueous solution thereof is alkaline. Noacid group is detected by analysis of titration curve. Namely thepresent substance is surely base.

Ultraviolet spectrum of free base crystal of SF-7 33 substance in anaqueous solution (0.102%) is shown in FIG. 4. No maximum absorption isobserved except end absorption. In the figure, numbers on abscissarepresent wave length (m and numbers on ordinate represent absorbancy.Infrared spectrum of its KBr tablet is shown in FIG. 5. Absorptionpeculiar to amino-glycosidic antibiotics is observed. In the figure,numbers on abscissa represent wave number per 1 cm. and numbers onordinate represent-transmission degree (percent).

The free base crystal of SF-733 substance is easily soluble in water buthardly soluble in methanol and very hardly soluble in ethanol. Furtherit is almost or entirely insoluble in n-propanol, n-butanol, acetone,ethyl acetate, benzene, petroleum either and chloroform.

The reactions of Molisch, anthrone, ninhydrin and fl-naphthol-sulfuricacid (aldopentose reaction) are positive but the reactions of anthroneunder defined condition [hexose, 6-deoxyhexose reactions (Methods ofCarbohydrate Chemistry, Vol. I, P. 490, 1963, Academic Press)], Fehling(cool), ferric chloride, Brady (reaction with2,4-dinitrophenylhydrazine), biuret, Sakaguchi, maltol are negative.Free base crystal of SF-733 substance is carbonized by addingconcentrated sulfuric acid to an aqueous solution thereof at a roomtemperature and reduces potassium permanganate solution at a roomtemperature.

'On thin layer chromatography of silica gel G (Merck & Co.) a singlespot is detected by ninhydrin coloration or sulfuric acid (carbonize byheating) coloration at almost original point with solvent system ofethanol-cone. ammonia water-water (8:1:1) and of 10% aqueous ammoniumacetate solution-methanol (1:1) at Rf value 0.5- 0.6 respectively. Onpaper bioautography using Bacillus subtilis, a single inhibitory spot isdetected at 8 cm. and 14.8 cm. from original point with solvent systemof n-butanol-pyridine-acetic acid-water (6:4:1z3) (descending, 4 days)and n-butanol saturated with water containing 2% p-toluene sulfonic acid(descending, 25 hours) respectively. In high voltage paperelectrophoresis, free base crystal of SF-733 substance moves cm. to thecathode and shows a single spot (3300' v, pH 1.8, 15 minutes, ninhydrincoloration, bioautography with Bacillus subtilis). On thin layerchromatography of silica gel G of N-acetyl derivative of free base ofSF-733, a single spot is detected by 10% sulfuric acid (carbonize byheating) coloration at Rf 0.40, 0.16, 0.50 and 0.70 with solvent systemof tert-butanol-acetic acid-water (2:1:1), n-butanol-aceticacid-water(3:1:1), n-butanol-pyridinewater (6:4:3) and 80% methanolrespectively.

Antibacterial activity of crystallized SF-733 substance andantibacterial spectrum thereof as shown in Table I are the same as thoseof amorphous SF-733 substance. Further their stability and toxicity arealso substantially the same.

The present invention will be explained more in detail by way offollowing examples.

EXAMPLE 1 Streptomyces thermoflavus SF-733 strain was inoculated to 15l. of a liquid medium (pH 7.0) containing glucose 2.5%, soybean meal3.5%, soluble vegetable protein 1.0% and NaCl 0.25% and shake-culturedin a jarfermenter at 28 C. for 3 days. 101. of culture filtrate(potency, 200 meg/ml.) obtained by filtering culture broth at pH 4.0 wasadjusted to pH 7.0 and applied to a column filled with 1 l. of AmberliteIRC 50 (NH; type, Rohm & Haas) to absorb active ingredient onion-exchange resin. After washing 'with water the column was eluted with0.5 N ammonia water. Active fractions were concentrated in vacuo andfreeze-dried. 5.9 g. of crude powder thus obtained was dissolved in 10ml. of water, applied to a column filled with 400 ml. of Dowel 1 x 2(OH- type, Dow Chemicals) and developed chromatographically with waterto give 250 ml. of active fraction which was concentrated in vacuo,whereby 2.1 g. of light yellow powder of SF-733 substance was obtained.2.0 g. of said powder was dissolved in 3 ml. of water, applied to acolumn filled with 100 ml. of Amberlite CG 50 (NH type) washed withwater and eluted with 0.2 N ammonia water. 400 ml. of active fractionwas collected, concentrated in vacuo and freeze-dried to give 600 mg.

of white powder of free base of SF-733 substance. This powder wasdissolved in about 5 ml. of water and concentrated to syrup and addedwith about 50 ml. of ethanol. The mother liquor together with whiteprecipitate thus formed was concentrated in vacuo to dryness. 650 mg. ofethanol-solvate like white powder was dissolved in 6.5 ml. of methanol.The solution became cloudy immediately after dissolution and crystalswere gradually separated. After tightly sealed and left alone at 30 C.over-night crystal was collected by means of glass filter and washedwith about 1 ml. of methanol. The crystal was held on calcium chlorideas a drying agent at a room temperature in vacuo and then dried onphosphorous pentoxide as a drying agent at 60 C. for 19 hours in vacuoto give 440 mg. of free base crystals of S F-733 substance. Yield: 73%

EXAMPLE 2 Streptomyces thermoflavus SF733 stain was inoculated to 40 l.of a liquid medium (pH 7.0) containing starch 4.0%, soybean meal 2.5%,wheat germ 1.0% and NaCl 0.25% and shake cultured in a jar-fermenter at35 C. for 2 days. 30 l. of culture filtrate (potency 350 mcg./ml.)obtained by filtering culture broth at pH 9.0 was adjusted to pH 7.0 andapplied to a column with filled 3 l. of Amberlite IRC 50 (Na type) toadsorb 97% of active ingredient on resin. After washing with water itwas eluted with 0.5 N HCl. 6.2 l. of active fraction was neutralizedwith Amberlite IR 45 (OH* type) and added with g. of active carbon understirring to adsorb almost all of active ingredient on active carbon.

This active carbon was washed with water and then eluted twice with each3 l. of 70% aqueous acetone adjusted pH to 2 with hydrochloric acid andthe eluate was neutralized, concentrated and freeze-dried. 8.4 g. ofcrude powder of SF-733 substance hydrochloride was dissolved in 12 ml.of water, applied to a column filled with 700 ml. of Dowex 1 x 2 (OH-type) and developed chromatographically with water. 650 ml. of activefraction was collected, concentrated in vacuo and freeze-dried to give4.2 g. of light yellow powder of free base of SF-733 substance. 4.1 g.of this powder was dissolved in a small amount 0 water and applied to acolumn filled with 100 ml. of Amberlite CG 50 (NH.;'' type) to give 3.4g. of white powder of free base of SF-733 substance.

Said powder was dissolved in 100 ml. of water, was concentrated in vacuowhile adding ethanol and finally water was distilled out by azeotropicdistillation. 3.8 g. of white ethanol-solvate like solid was disolved in38 ml. of methanol and tightly sealed and left alone at a roomtemperature over-night to separate crystal. The crystal was collected byfiltration, washed with 10 ml. of methanol and dried by a methodsubstantially similar to Example 1 to give 2.4 g. of free base crystalsof SF-733 substance. Yield 71%.

EXAMPLE 3 To 10 l. of the culture broth obtained in Example 1 Hyflosupercell was added as a filter aid and mycelia were removed by filtration.8 1. of this filtrate was adjusted to pH 8.5-9.5 and active carbon in anamount (80 g.) corresponding to 1% of the filtrate was added and stirredfor 30 minutes whereby almost all of active ingredient was adsorbed toactive carbon. The active carbon was collected by filtration, washedwith water, suspended in 1.2 l. of 70% aqueous methanol, adjusted pH to2.0 with 4 N sulfuric acid and stirred for 30 minutes to extract activeingredient. The filtrate after removing active carbon by filtration wasconcentrated in vacuo and added with acetone whereby 28 g. of crudepowder containing SF-733 substance was obtained. It was dissolved in 35ml. of water, adjusted to pH 4.0-4.5 with 4 N sulfuric acid, applied toa column filled with 35- g. of active carbon powder and eluted wtih 0.03N sulfuric acid. 300 ml. of active fraction was collected, adjusted topH 6.0

1 1 with Amberlite IR 45 (OH type), concentrated in vacuo andfreeze-dried to give 6.5 g. of crude powder of SF-733 substance sulfate.

We claim:

1. A process for the production of a new antibiotic CF-733 substancewhich comprises cultivating a strain of Streptomyces thermoflavus ATCC21294 and mutants thereof which produce SF-733 in an aqueous nutrientmedium containing assimilable nitrogenand carbonsources under submergedaerobic condition to accumulate SF-733 substance in the medium and thenrecovering said SF-733 substance.

2. A process for the production of free base crystal of 81 -733substance which comprises cultivating SF-733 strain of Streptomycesthermoflavus ATCC 21294 and mutants thereof which produce SF-733 in anaqueous nutrient medium containing assimilable nitrogenandcarhon-sources under submerged aerobic condition to accumulate amorphousfree base of SF-733 substance in the medium, collecting said amorphousfree base of SF-733 substance, dissolving it in Water, drying theaqueous solution together with ethanol to solid, dissolving the solid inmethanol and recovering free base crystal of SF-733 substance separatedfrom methanol solution.

3. Process for the production of free base crystal of SF-733 substancewhich comprises cultivating SF-733 strain of Streptomyces thermoflavusATCC 21294 and mutants thereof which produce SF-733 in an aqueousnutrient medium containing assimilable nitrogen source and carbon sourceunder submerged aerobic condition to accumulate amorphous free base ofSF-733 substance in the medium, collecting said amorphous free base ofSF- 733 substance, dissolving it in water, concentrating the solution todryness while adding ethanol and distilling out water by azeotropicdistillation to give white solid SF-733 substance in the form ofethanol-solvate, dissolving said ethanol-solvate substance in methanol,leaving the methanol solution alone to crystallization and recoveringthe free base crystal of SF-733 substance thus formed from methanolsolution.

4. A process for the production of free base crystal of SF-733 substancewhich comprises cultivating SF-733 strain of Streptomyces flavus in anaqueous nutrient medium containing assimilable nitrogen source andcarbon source under submerged aerobic condition to accumulate amorphousfree base of SF-733 substance in the medium, collecting said amorphousfree base of SF-733 substance, dissolving it in water, concentrating thesolution to syrup, adding ethanol to the syrup to form precipitate,concentratin the syrup containing the precipitate to dryness whilefurther adding ethanol and distilling out water by azeotropicdistillation to give white solid ISF-733 substance in the form ofethanol-solvate, dissolving said ethanol-solvate substance in methanol,leaving the methanol solution alone to crystallization and recoveringthe free base crystal of SF-733 substance thus formed from ethanolsolution.

References Cited UNITED STATES PATENTS 3,616,243 10/1971 Kawaji et a1.l--80 R JOSEPH M. GOLIAN, Primary Examiner US. Cl. X.R. 260-2l0 AB

